The normal physiology of the lens is poorly understood. Because the lens is avascular, it must solve the problem of transporting nutrients into its interior and removing waste products in a fashion not required of most other tissues. Several investigators have proposed that an intrinsic circulation generated by the lens itself and driven by active processes taking place near the surface of the lens where oxygen and nutrients are most available accomplishes this. There is growing if indirect evidence that the lens indeed possesses such a circulation and that it varies in detail amongst different species. Our research focuses on the role of aquaporin zero (formerly called MIP, the major intrinsic protein of the lens). We have found that AQP0 water permeability can be more that doubled by decreasing pH or calcium concentration in the physiological concentration ranges of these ions found in the lens. We therefore propose that such regulation of circulation would be entirely suitable to lens which must operate on a very tight energy budget and cannot afford to use complex energy requiring regulatory mechanisms. We have found that histidines in the external loops of AQP0 are essential for the pH regulation function and that calmodulin mediates the calcium regulation. We have managed to make mutant proteins deficient in both types of regulation. Most interestingly we have been able to confer the property of pH regulation on Aqp1, a member of the aquaporin family, which normally lacks. This gives us confidence that we are on the right track toward a molecular understanding of this phenomenon. In this proposal we will investigate the molecular mechanisms by which AQP0 accomplishes its changes of water permeability and by using transgenic knock ins of AQP0 mutants deficient in specific modes of regulation we will investigate the physiological role of AQP0 water permeability regulation in the lens.